The effectiveness of vital stains to assess the viability of hookworm eggs depends on the permeability of eggshells. The eggshells may not be permeable immediately after inactivation, and this can lead to over-estimation of viable eggs in a sample. In this study, heat-inactivated eggs of hookworm (Ancylostoma caninum) were used to evaluate the time required for eggshells to become permeable to three vital stains (eosin Y, methyl red, and methylene blue). The results of this study showed that heat-inactivated eggs incubated for 1 h at room temperature (24 ± 1 °C) contain 32-53% non-viable eggs or permeable eggshells. The numbers of non-viable eggs increased to 88-94% after 6 h of incubation. The accurate viability assessment was achieved after 12 h of incubation at room temperature. Vital stains did not play a significant (P > 0.05) role in reducing the false viability. This study provides information that can aid in the accurate detection of viable hookworm eggs from environmental samples comprised of mixed populations that are viable and non-viable with permeable or impermeable eggshells.